Amplification of nucleic acids and analysis of the resulting amplification products are used in cloning, sequencing, genotyping, and gene expression. Multiple techniques for nucleic acids amplification have been developed, such as polymerase chain reaction (PCR), strand displacement amplification, and transcription-based amplification. Unfortunately, current methods are non-specific with respect to preserving the sequence information at the ends of nucleic acids. For example, in PCR, the binding of a forward and a reverse oligonucleotide to a target nucleic acid will produce an amplicon with a sequence corresponding to the sequence of the target nucleic acid between the forward and reverse oligonucleotides. In many instances, the forward and reverse oligonucleotides may bind at a region other than the end of the target nucleic acid, thereby producing an amplicon shorter than the target nucleic acid (e.g., losing the sequence information at the ends of the target nucleic acid). However, the sequence information at the ends of nucleic acid fragments (e.g., cell-free nucleic acid fragments) can be very useful in disease prognostics and diagnostics. Accordingly, the present disclosure provides methods for enriching and amplifying target nucleic acids in a sample while preserving the sequence information at the ends of the target nucleic acids.
Amplification of nucleic acids and subsequent analysis of the resulting amplification products (e.g., amplicons) can be performed in a number of molecular assays including molecular cloning, sequencing, genotyping, and gene expression. Amplification can be particularly useful where samples contain relatively small quantities of starting template material (e.g., nucleic acids). Multiple reports indicate that circulating cell-free DNA (cfDNA) in plasma can be non-randomly fragmented. Extremely low concentration of cfDNA in plasma (10-5000 genomes/ml) can make the study and detection of the fragmentation pattern difficult. While techniques such as whole genome amplification (WGA) have been developed to amplify small quantities of nucleic acids, the amplicons can be shorter than the template fragments from which the amplicons are produced. In some cases, nucleotides at the ends of the starting template nucleic acid fragments are not copied (e.g., these techniques can fail to preserve sequence information at the ends of template nucleic acid fragments), and are not useful for determining nucleic acid fragmentation patterns. The present disclosure provides compositions and the methods for the amplification of nucleic acid molecules (e.g., cell-free DNA molecules) while preserving sequence information at the ends of the molecules. Furthermore, the present disclosure provides embodiments for determining the nucleic acid fragmentation pattern following amplification where the sequence information at the ends of the nucleic acid molecules are preserved.